Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405, USA.

2

Department of Mathematics, University of California, Davis, California 95616, USA.

Abstract

Intracellular cargo transport relies on myosin Va molecular motor ensembles to travel along the cell's three-dimensional (3D) highway of actin filaments. At actin filament intersections, the intersecting filament is a structural barrier to and an alternate track for directed cargo transport. Here we use 3D super-resolution fluorescence imaging to determine the directional outcome (that is, continues straight, turns or terminates) for an ∼10 motor ensemble transporting a 350 nm lipid-bound cargo that encounters a suspended 3D actin filament intersection in vitro. Motor-cargo complexes that interact with the intersecting filament go straight through the intersection 62% of the time, nearly twice that for turning. To explain this, we develop an in silico model, supported by optical trapping data, suggesting that the motors' diffusive movements on the vesicle surface and the extent of their engagement with the two intersecting actin tracks biases the motor-cargo complex on average to go straight through the intersection.

(a) Schematic of granule (yellow) transport by myoVa ensembles through the actin cortex. Transport from A to B (red arrow) presents a number of physical and directional challenges. (b) Zoom in from A. Multiple-myoVa motors (black) are bound and free to diffuse (dashed arrows) on the surface of a lipid-bound cargo (yellow). One or more motors at different regions on the cargo surface (green) can simultaneously engage a single filament. In this illustration two sets of motors interact with individual actin filaments (blue, magenta) and undergo a tug-of-war to determine the direction of cargo transport. (c) STORM image of 3D actin network and intersections created by stringing actin between 3 μm beads; Z-position shown in colour. Scale bar: 2,000 nm. (d) Time sequence of liposome (yellow) transported by myoVa motors turning (red dashed arrow) at actin filament intersection. Actin Z-position is defined by the colour bar in B. Scale bar: 500 nm. (e) Liposome continuing straight through a different 3D intersection. Scale bar: 500 nm.

(a) To scale schematic of a 3D suspended actin intersection encountered by a motor–cargo complex where the approach angle (α) and filament separation (d) are defined. An α of 0° has the centre of the motor–cargo complex vertically above the original filament it is travelling on, which is the same side the intersecting filament is on. d is the centre-to-centre distance between the two intersecting filaments at the point of the intersection. (b) Polar plot predicting whether or not a motor–cargo complex physically interacts with the intersecting filament as a function of d (0–250 nm) and α (0°–360°). To translate this plot into 3D spatial relations between the intersecting filaments and the motor–cargo complex, the illustration of the approach angle (α) in a is used as the point of reference. The originally bound filament on which the motor–cargo complex is travelling on comes in and out of the figure at the graph's origin. The intersecting filament is horizontally in the plane of the figure at a filament separation, d, above the origin with α defined as in a. The magenta line is the predicted spatial boundary at which combinations of d and α determine whether or not the motor–cargo complex can physically interact with the intersecting filament. To scale examples of interaction and non-interaction geometries are illustrated at their respective d and α. The coloured triangles for these examples are identified on the polar plot. (c) Fractional probability plot of motor-complex directional outcomes for experimental (solid bars, n=94) and modelled (slashed bars) data at 3D intersections with predicted interaction geometries. Directional outcomes at 2D experimental intersections (vertical striped bars, n=96) where the motor–cargo complex approached the intersecting filament on the bottom filament. Of the turning outcomes in the 3D experiments, left hand turns occurred 52% of the time with right hand turns the other 48%. For the model the left and right turn probability was equal. The model also predicts (inset) that the intersection directional outcomes are independent of the polarity of the intersecting filament. Error bars are s.d. from six simulations.

Heatmap of straight-to-turn ratio at 3D intersections plotted in polar coordinates as in as a function of approach angle (α) and filament separation (d).

Due to the symmetry in the system (see text), all motor–cargo approach angles between 180 and 360° were mirrored onto 0–180°(). Straight-to-turn ratio from the experimental data (left half, n=103) and modelled data (right half, n=4,000) were sorted by α and d into four interaction regimes and one non-interaction regime with the number of events for the experimental results in parentheses. The straight-to-turn ratio in each regime is colour coded (see colour bar). In both the experimental and modelled data, straight outcomes were predominant in all regimes and highest in the non-interaction regimes.

Measurement of motor–cargo complex force production and model simulations.

(a) Schematic of laser trap experiments. (b) Displacement and force trace of motor–cargo complex moving against the restoring force of the laser trap (trap stiffness=0.019±0.004 pN nm−1) (Methods section). Saw tooth pattern arises from motors pulling the cargo until reaching a stall force (that is, plateau) and then detaching only to be repeated multiple times. (c) Histogram of peak force before reversal or detachment for cargo transported by multi-motors (green) and a single motor (blue). (d) Model predicted relative binding rate (red colour bar) for an additional myoVa motor (yellow) to available actin monomers, given 1 (top), 2 (middle) or 3 (bottom) previously bound motors. Relative rate of binding for a new motor decreases with each additionally bound motor. (e–h) Illustration of model result showing how three motors (yellow) transporting lipid-bound cargo (blue) along an actin filament (green) can go straight even when an intersecting filament (red) acts as a physical barrier at the given approach angle (α) and filament separation (d). (f) Single-motor engages intersecting actin, resulting in a tug-of-war between motor ensembles attached to both filaments. (g) Stochastic motor detachment and binding leads to cargo repositioning. (h) With additional repositioning, the intersecting filament no longer acts as a physical barrier, thus allowing the motors to continue straight along the original filament.